Surface proteins from gram-positive bacteria having highly conserved motifs and antibodies that recognize them

ABSTRACT

Isolated peptide sequences and proteins containing these sequences are provided which are useful in the prevention and treatment of infection caused by Gram-positive bacteria. The peptide sequences have been shown to be highly conserved motifs in the surface proteins of Gram-positive bacteria, and these consensus sequences include amino acid sequences such as LPXTG (SEQ ID NO:13), ALKTGKIDIIISGMTSTPERKK (SEQ ID NO:14), VEGAWEKPVAEAYLKQN (SEQ ID NO:15), and EYAGVDIDLAKKIAK (SEQ ID NO:16). By virtue of the highly conserved regions, the sequences and the proteins including these sequences can be utilized to generate antibodies which can recognize these highly conserved motifs and the proteins containing them and thus be useful in the treatment or prevention of a wide range of infections caused by Gram-positive bacteria.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No. 60/289,132, filed May 8, 2001.

FIELD OF THE INVENTION

The present invention relates in general to surface-located proteins from gram-positive bacteria, and in particular to a group of proteins that contain highly conserved sequence motifs. In addition, the invention relates to polyclonal and monoclonal antibodies which can recognize these proteins and which can recognize the conserved motifs. Further, the invention relates to the use of the proteins, conserved motifs and antibodies generated thereto in compositions and methods used to treat or prevent infections and other pathogenic conditions caused by a wide-array of gram-positive bacteria.

BACKGROUND OF THE INVENTION

Bacterial surface proteins of gram-positive bacteria are known to be important during the infection process since they mediate bacterial attachment to host tissues, and/or interact with the host immune system. For example, in the gram-positive bacteria Staphylococcus aureus, several of these proteins have been well characterized and were found to bind extracellular matrix proteins such as collagen, fibronectin, fibrinogen, as well as immunoglobulin G. These binding proteins include fibronectin binding proteins such as disclosed in U.S. Pat. Nos. 5,175,096; 5,320,951; 5,416,021; 5,440,014; 5,571,514; 5,652,217; 5,707,702; 5,789,549; 5,840,846; 5,980,908; and 6,086,895; fibrinogen binding proteins such as disclosed in U.S. Pat. Nos. 6,008,341 and 6,177,084; and collagen binding proteins as disclosed in 5,851,794 and 6,288,214; all of these patents incorporated herein by reference.

Previous studies have shown that the collagen and fibronectin binding proteins have been shown to contribute to the virulence of S. aureus in animal models. In addition, immunization of mice with certain of these binding protein has been shown in some cases to provide protection from septic death due to S. aureus. However, in some cases, certain formulations based on bacterial proteins from specific gram-positive bacteria such as S. aureus were not always effective in treating patients, and moreover these formulations will generally be species specific and thus do not generally afford protection against infection from a variety of gram-positive bacteria. Accordingly, it is very important to develop ways of locating surface proteins which will be utilized effectively in methods of treating or preventing infection, and in particular it is highly desirable to develop methods of treatment which can be utilized in a broad-based application to treat or prevent a wide variety of infections caused by gram-positive bacteria.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide methods for isolating proteins from gram-positive bacteria which can be utilized in methods of treating or preventing a wide range of infections caused by gram-positive bacteria.

It is another object of the present invention to provide surface proteins from gram-positive bacteria that have highly conserved sequence motifs near their carboxyl termini which can be utilized to generate antibodies that will be protective against a wide variety of gram positive bacteria.

It is further an object of the present invention to provide a method of generating an immune response to a wide variety of gram-positive bacteria by administering an immunogenic amount of an isolated peptide sequence which is highly conserved in gram positive bacteria or by administering proteins which include these highly conserved sequence motifs.

It is a further object of the present invention to provide a vaccine for treating or preventing infection from gram-positive bacteria which comprises an isolated peptide sequence which is highly conserved in gram positive bacteria or a protein which includes one or more of these highly conserved sequence motifs in an amount effective to generate an immune response to said peptides or proteins.

It is still further an object to provide compositions for treating or preventing an infection from gram-positive bacteria which comprise an isolated peptide sequence which is highly conserved in gram positive bacteria or a protein which includes one or more of these highly conserved sequence motifs and a pharmaceutically acceptable vehicle, carrier or excipient.

It is still further an object of the present invention to provide isolated antibodies which recognize these highly conserved sequence motifs or proteins which contain said motifs, and to utilize these antibodies in treating or preventing infection caused by a broad range of gram-positive bacteria.

It is an additional object of the present invention to provide diagnostic kits which can utilize the conserved sequences, proteins, and/or antibodies in accordance with the invention in order to diagnose and identify infections caused by gram-positive bacteria.

These and other objects are provided by virtue of the present invention which comprises the identification, isolation, and/or purification of highly conserved amino acid sequences from gram positive bacteria and proteins which contain said sequences, and the use of these sequences and/or proteins to treat or prevent infections caused by a wide range of gram-positive bacteria. In addition, the invention comprises monoclonal and polyclonal antibodies which recognize these sequences and proteins, as well as vaccines and other pharmaceutical compositions which utilize these peptide sequences and proteins, and methods of eliciting an immune response against a broad range of gram positive bacteria by administering the peptides and/or proteins to a human or animal in an amount effective to generate an immune response. The sequences and proteins of the present invention can thus be used in methods or achieving passive or active immunity in patients so as to treat or prevent a wide range of infections caused by gram-positive bacteria.

These embodiments and other alternatives and modifications within the spirit and scope of the disclosed invention are described in, or will become readily apparent from, reference to the detailed description of the preferred embodiments provided herein below.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

FIG. 1 is a depiction of ClustalW multiple sequence alignment of the amino acid sequences of the surface proteins in accordance with the invention which have been characterized as the cell division group (or group 1) from 6 Gram-positive bacteria, shown from top to bottom as saur, S. aureus; sepi, S. epidermidis; smut, S. mutans; spne, S. pneunomiae; efae; E. faecalis; and spyo, S. pyogenes, which are identified, respectively, as SEQ ID NOS 1-6. In the drawing figure, the dark-shaded regions represent highly conserved residues, and light-shaded regions represent relatively well-conserved residues.

FIG. 2 is a depiction of ClustalW multiple sequence alignment of the amino acid sequences of the surface proteins in accordance with the invention which have been characterized as the amino acid transporter group (or group 2) from 6 Gram-positive bacteria, shown from top to bottom as spyo, S. pyogenes; spne, S. pneunomiae; smut, S. mutans; efae, E. faecalis; saur, S. aureus; and sepi, S. epidermidis; and which are identified, respectively, as SEQ ID NOS 7-12. In the drawing figure, the dark-shaded regions represent highly conserved residues, and light-shaded regions represent relatively well-conserved residues.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, the present inventors have isolated novel surfaces proteins from gram positive bacteria that are characterized in that they contain highly conserved sequences which can be utilized in the identification and isolation of surface proteins from gram positive bacteria, and which can be used to generate antibodies which will recognize said highly conserved sequences and/or the surface proteins containing said sequences. In particular, these novel proteins containing their unique highly conserved sequences were obtained in accordance with the invention using an algorithm the present inventors devised for reviewing publicly available sequence information regarding Gram-positive bacteria so as to identify and/or isolate and purify highly conserved regions in the genome and the proteins which contain those highly conserved regions. In the identification and isolation process of the invention, numerous genomes from Gram-positive bacteria are selected, and in a suitable example, genomes of six Gram-positive bacteria, namely Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus Faecalis, Streptococcus pyogenes, Streptococcus pneumoniae, and Streptococcus mutans, all of which are important human pathogens, were selected and subject to the present identification process. The genomes of four S. aureus strains were publicly available at the time of the analysis and were all included in the process to identify conserved regions of the genome and/or proteome.

In the specific example, the S. aureus genome sequences were obtained from the websites of The Institute for Genomic Research (TIGR) (strain COL), The Sanger Center (a methicillin resistant strain and a methicillin sensitive strain), and University of Oklahoma's Advanced Center for Genome Technology (OU-ACGT) (strain 8325). The genome sequences of E. faecalis (strain V583), S. epidermidis (strain RP62A) and S. pneumoniae Type 4 were obtained from TIGR, and the sequences of S. mutans and S. pyogenes (group A) were from OU-ACGT.

In one preferred process, the identification steps or “data mining” was performed using a combination of software developed by the inventors, Glimmer2 from TIGR and stand-alone BLAST from the National Center for Biotechnology Information. The system was set up on a Silicon Graphics machine running IRIX6.5. In the preferred process of the present invention, an algorithm is used which consists of the following steps: (1) process each sequence file which usually contains multiple contigs into individual files each of which consists one contig; (2) predict genes based on the sequencing; (3) add a unique identification tag to each predicted gene so that genes from different organisms can be put into one single database; (4) extract genes from each genome; (5) translate each gene into its amino acid sequences; (6) form a blast searchable database of the protein sequences; and (7) perform a blast search of the database to find proteins that contain the desired conserved motif such as the LPXTG (SEQ ID NO: 13) motif wherein X can be any amino acid.

After LPXTG-containing proteins were identified, they were collected into a subset and used to establish a separate blast searchable database. Each protein in this subset was blasted against each other as well as to the large protein database to identify LPXTG-containing proteins that are conserved among these organisms. In the analysis of the LPXTG-containing proteins, two groups were located as discussed further below. Members in each group exhibited substantial overall sequence homology with each other as can be seen from Tables 1 and 2.

TABLE 1 Percentage amino acid sequence similarities among the cell division group (group 1) from 6 Gram-positive bacteria. S. S. S. E. epider- pneu- pyo- S. S. faecalis midis moniae genes mutans aureus E. faecalis 100 55 66 41 67 56 S. epidermidis 100 52 41 53 90 S. pneumoniae 100 45 78 51 S. pyogenes 100 44 41 S. mutans 100 52 S. aureus 100

TABLE 2 Percentage amino acid sequence similarities among the amino acid transporter group (group 2) from 6 Gram-positive bacteria. S. S. S. pneu- pyo- S. epider- S. E. moniae genes mutans midis aureus faecalis S. pneumoniae 100 81 91 51 51 69 S. pyogenes 100 81 48 48 68 S. mutans 100 51 51 67 S. epidermidis 100 87 49 S. aureus 100 49 E. faecalis 100

In addition, after multiple sequence alignment, there are stretches of completely identical sequences in each group, as shown in FIGS. 1 and 2. Moreover, a homology search with known genes indicated that the first group (SEQ ID NOS 1-6 of FIG. 1) appeared to be a novel group of proteins that belonged to a family of cell division proteins, while the second group (SEQ ID NOS 7-12 of FIG. 2) appeared to be characterized as a family of amino acid transporters. However, none of the proteins in the two groups has been described for the organisms that were analyzed, and therefore they are novel for these bacteria.

In addition, each protein in the two groups was examined for the presence of signal peptide through the SignalP mail server at Center for Biological Sequence Analysis, the Technical University of Denmark. Each was predicted to contain a signal peptide at the proper position, which appeared to confirm that these are surface proteins. In general, cell division proteins and amino acid transporters are important proteins for bacteria survival in vitro and in vivo. The fact that these proteins exhibit such high-level sequence conservation among the organisms suggests that they perform conserved functions, and it is clear that similar surface proteins are present in other Gram-positive bacteria which will also be characterized by the conserved regions in accordance with the present invention.

In addition to the sequence motif LPXTG which was discussed above, the present inventors uncovered 3 additional novel peptide sequences motifs that were conserved in the proteins identified using the method as described above. In particular, these conserved regions have the amino acid sequences identified as “SA-1”: ALKTGKIDIIISGMTSTPERKK (SEQ ID NO:14); “SA-2”: VEGAWEKPVAEAYLKQN (SEQ ID NO:15), and “SA-3”: EYAGVDIDLAKKIAK (SEQ ID NO:16). The peptide sequences were selected from 3 regions in a Staphylococcus aureus protein that belongs to one ABC transporter group. Each region is highly conserved among the 6 Gram-positive bacteria examined (Enterococcus faecalis, Staphylococcus epidernidis, Streptococcus pyogenes, Streptococcus mutans, Streptococcus pneumoniae, and Staphylococcus aureus). Also, in order to increase the chance that the sequences will be exposed on the surface, we limited the selection of the sequences to hydrophilic regions using the method of Kyte and Doolittle.

In accordance with the present invention, these specific peptides may be obtained in any of a number of suitable ways well known in the art to generate peptides, and similarly, proteins containing these peptides may be obtained through physical isolation and/or separation methods from actual bacteria, or through conventional methods of protein synthesis. In the present case, one suitable method for preparing the peptides of the invention is through synthesis using an Advanced Chem Tech 396 multiple peptide synthesizer, using Fmoc chemistry and activation with HBTU. After cleavage from the resin, peptides can be purified by reverse-phase chromatography on a Waters Delta-Pak C18 column, eluted with gradient of acetonitrile in 0.1% trifluoroacetic acid/water. The purity of the peptides obtained in this fashion has been further confirmed by mass spectrometry analysis, and the peptide-KLH conjugation with EDC. The carrier protein KLH and the peptides (1:1 by weight) were coupled using EDC (Pierce) for 2 hours at room temperature. The reaction mixture is subjected to a desalting column pre-equilibrated with the purification buffer (0.083 M sodium phosphate, 0.9 M NaCl, pH 7.2). The conjugated peptides were eluted with the purification buffer and 0.5 ml fractions were collected. Each fraction was measured for its absorbance at 280 nm and the fractions containing the conjugate were pooled.

Accordingly, in accordance with the present invention, there are provided isolated amino acid sequences, namely ALKTGKIDIIISGMTSTPERKK (SEQ ID NO:14); VEGAWEKPVAEAYLKQN (SEQ ID NO:15), and EYAGVDIDLAKKIAK (SEQ ID NO:16), which are highly conserved regions in surface proteins from Gram-positive bacteria which can be utilized to generate antibodies that can recognize these sequences and which thus can be utilized in methods of treating or preventing a wide range of Gram-positive bacteria that will have proteins containing these sequences. In addition, it is contemplated that proteins from Gram-positive bacteria that contain these conserved sequences may also be isolated and/or purified, and may also be used to generate antibodies which recognize these proteins and which can be utilized in methods of treating or preventing infection caused by Gram-positive bacteria.

In accordance with the invention, the antibodies generated by immunization with either the conserved sequences described above or proteins containing these sequences may be either monoclonal or polyclonal, and may be prepared in any of a number of conventional ways well known to those of ordinary skill in the art. For example, monoclonal antibodies in accordance with the present invention may be produced, e.g., using the method of Kohler and Milstein (Nature 256:495-497, 1975), or other suitable ways known in the field, and in addition can be prepared as chimeric, humanized, or human monoclonal antibodies in ways that would be well known in this field. Still further, monoclonal antibodies may be prepared from a single chain, such as the light or heavy chains, and in addition may be prepared from active fragments of an antibody which retain the binding characteristics (e.g., specificity and/or affinity) of the whole antibody. By active fragments is meant an antibody fragment which has the same binding specificity as a complete antibody which recognizes and binds to the peptide sequences or the proteins of the present invention, and the term “antibody” as used herein is meant to include said fragments. Additionally, antisera prepared using monoclonal or polyclonal antibodies in accordance with the invention are also contemplated and may be prepared in a number of suitable ways as would be recognized by one skilled in the art.

As indicated above, antibodies which recognize the conserved sequences, or proteins containing these sequences, as set forth above, may be prepared in a number of suitable ways that would be well known in the art, such as the well-established Kohler and Milstein method described above which can be utilized to generate monoclonal antibodies. In one such method, mice are injected intraperitoneally once a week for a prolonged period with a antigen comprising a purified recombinant peptide or protein in accordance with the invention, followed by a test of blood obtained from the immunized mice to determine reactivity to the purified antigen. Following identification of mice suitably reactive to the antigen, lymphocytes isolated from mouse spleens may be fused to mouse myeloma cells to produce hybridomas positive for the antibodies against the peptides and/or proteins of the invention which are then isolated and cultured, following by purification and isotyping.

In order to generate monoclonal antibodies in accordance with the invention, it is thus preferred that these be generated using recombinantly prepared peptide sequences or proteins using conventional methods well known in the art. For example, one such method employs the use of E. coli expression vector pQE-30 as an expression vector for cloning and expressing recombinant proteins and peptides. In this method, PCR is used to amplify DNA coding for the peptide sequences of the invention, and a suitable E. coli expression vector such as PQE-30 (Qiagen) is used to allow for the expression of a recombinant fusion protein having the appropriate sequences. The cells containing these fusion proteins may be harvested, and the peptides of the invention may be eluted suing suitable buffer solutions. The peptides can then be subject to further purification steps, e.g., put through an endotoxin removal process, and the appropriate peptides obtained in this fashion may then be utilized to elicit an immune response and generate antibodies in accordance with the invention.

As indicated above, although production of antibodies using recombinant forms of the peptides or proteins of the invention is preferred, antibodies may be generated from natural isolated and purified proteins or peptides as well, and monoclonal or polyclonal antibodies can be generated using the natural peptides or proteins or active regions in the same manner as described above to obtain such antibodies. Still other conventional ways are available to generate the antibodies of the present invention using recombinant or natural purified peptides or proteins or its active regions, as would be recognized by one skilled in the art.

As would be recognized by one skilled in the art, the antibodies of the present invention may also be formed into suitable pharmaceutical compositions for administration to a human or animal patient in order to treat or prevent an infection caused by Gram-positive bacteria. Pharmaceutical compositions containing the antibodies of the present invention, or effective fragments thereof, may be formulated in combination with any suitable pharmaceutical vehicle, excipient or carrier that would commonly be used in this art, including such as saline, dextrose, water, glycerol, ethanol, other therapeutic compounds, and combinations thereof. As one skilled in this art would recognize, the particular vehicle, excipient or carrier used will vary depending on the patient and the patient's condition, and a variety of modes of administration would be suitable for the compositions of the invention, as would be recognized by one of ordinary skill in this art. Suitable methods of administration of any pharmaceutical composition disclosed in this application include, but are not limited to, topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal and intradermal administration.

For topical administration, the composition is formulated in the form of an ointment, cream, gel, lotion, drops (such as eye drops and ear drops), or solution (such as mouthwash). Wound or surgical dressings, sutures and aerosols may be impregnated with the composition. The composition may contain conventional additives, such as preservatives, solvents to promote penetration, and emollients. Topical formulations may also contain conventional carriers such as cream or ointment bases, ethanol, or oleyl alcohol.

Additional forms of antibody compositions, and other information concerning compositions, methods and applications with regard to other surface proteins will generally also be applicable to the present invention, including those antibodies and compositions as disclosed, for example, in U.S. Pat. No. 6,288,214 (Hook et al.), incorporated herein by reference. Similarly, other forms of antibody compositions, and other information concerning compositions, methods and applications with regard to other surface proteins and peptides which will also be applicable to the present invention are disclosed in U.S. Ser. No. 09/810,428, filed Mar. 19, 2001, incorporated herein by reference; and U.S. Ser. No. 09/386,962, filed Aug. 31, 1999, incorporated herein by reference.

The antibody compositions of the present invention may also be administered with a suitable adjuvant in an amount effective to enhance the immunogenic response against the conjugate. For example, suitable adjuvants may include alum (aluminum phosphate or aluminum hydroxide), which is used widely in humans, and other adjuvants such as saponin and its purified component Quil A, Freund's complete adjuvant, RIBBI adjuvant, and other adjuvants used in research and veterinary applications. Still other chemically defined preparations such as muramyl dipeptide, monophosphoryl lipid A, phospholipid conjugates such as those described by Goodman-Snitkoff et al. J. Immunol. 147:410-415 (1991) and incorporated by reference herein, encapsulation of the conjugate within a proteoliposome as described by Miller et al., J. Exp. Med. 176:1739-1744 (1992) and incorporated by reference herein, and encapsulation of the protein in lipid vesicles such as Novasome™ lipid vesicles (Micro Vescular Systems, Inc., Nashua, N.H.) may also be useful.

In any event, the antibody compositions of the present invention will thus be useful for treating or preventing infections caused by gram-positive bacteria and/or in reducing or eliminating the binding of gram-positive bacteria to host cells and/or tissues.

In accordance with the present invention, isolated and/or purified conserved amino acid sequences such as SEQ ID NOS 14-16 are provided which can be utilized in methods of treating or preventing a Gram-positive bacterial infection. Accordingly, in accordance with the invention, nucleic acids are provided which encode the peptide sequences of the invention and which encode the proteins which contain these conserved sequences, or degenerates thereof.

As indicated above, in accordance with the present invention, methods are provided for preventing or treating a Gram-positive bacterial infection which comprise administering an effective amount of an antibody to the peptides or proteins identified above in amounts effective to treat or prevent the infection. In addition, the antibodies in accordance with the invention are particularly effective against a wide range of Gram-positive bacteria because they can recognize conserved peptide sequences, and/or proteins containing these sequences therein, which will be found in the wide range of gram-positive bacteria that commonly cause infection in human or animal patients.

Accordingly, in accordance with the invention, administration of the antibodies of the present invention in any of the conventional ways described above (e.g., topical, parenteral, intramuscular, etc.), and will thus provide an extremely useful method of treating or preventing Gram-positive bacterial infections in human or animal patients. By effective amount is meant that level of use, such as of an antibody titer, that will be sufficient to either prevent adherence of the gram-positive bacteria, or to inhibit binding of the bacteria to host cells, and thus will be useful in the treatment or prevention of a gram-positive bacterial infection. As would be recognized by one of ordinary skill in this art, the level of antibody titer needed to be effective in treating or preventing a particular Gram-positive infection will vary depending on the nature and condition of the patient, and/or the severity of the pre-existing infection.

In addition to the use of the present antibodies to treat or prevent Gram-positive bacterial infection, the present invention contemplates the use of these antibodies in a variety of ways, including the detection of the presence of gram-positive bacteria to diagnose a bacterial infection, whether in a patient or on medical equipment which may also become infected. In accordance with the invention, a preferred method of detecting the presence of such infections involves the steps of obtaining a sample suspected of being infected by one or more Gram-positive bacteria species or strains, such as a sample taken from an individual, for example, from one's blood, saliva, tissues, bone, muscle, cartilage, or skin. While adequate diagnostic tests can be performed using the sample itself, it is also possible to perform more complex tests which utilize the DNA of the sample. In these diagnostic tests, the cells can then be lysed, and the DNA extracted, precipitated and amplified. Following isolation of the sample, diagnostic assays utilizing the antibodies of the present invention may be carried out to detect the presence of Gram-positive bacteria, and such assay techniques for determining such presence in a sample are well known to those skilled in the art and include methods such as radioimmunoasssay, Western blot analysis and ELISA assays. In general, in accordance with the invention, a method of diagnosing a Gram-positive bacterial infection is contemplated wherein a sample suspected of being infected with such bacteria has added to it an antibody in accordance with the present invention, and a Gram-positive bacterial infection will be indicated by antibody binding to the appropriate proteins or peptides in the sample.

Accordingly, antibodies in accordance with the invention may be used for the specific detection of gram-positive bacterial or surface proteins, for the prevention of infection from Gram-positive bacteria, for the treatment of an ongoing infection, or for use as research tools. The term “antibodies” as used herein includes monoclonal, polyclonal, chimeric, single chain, bispecific, simianized, and humanized or primatized antibodies as well as Fab fragments, such as those fragments which maintain the binding specificity of the antibodies to the peptides and/or proteins of the present invention, including the products of an Fab immunoglobulin expression library. Accordingly, the invention contemplates the use of single chains such as the variable heavy and light chains of the antibodies as set forth above. Generation of any of these types of antibodies or antibody fragments is well known to those skilled in the art.

Any of the above described antibodies may be labeled directly with a detectable label for identification and quantification of Gram-positive bacteria. Labels for use in immunoassays are generally known to those skilled in the art and include enzymes, radioisotopes, and fluorescent, luminescent and chromogenic substances, including colored particles such as colloidal gold or latex beads. Suitable immunoassays include enzyme-linked immunosorbent assays (ELISA).

Alternatively, the antibody may be labeled indirectly by reaction with labeled substances that have an affinity for immunoglobulin. The antibody may be conjugated with a second substance and detected with a labeled third substance having an affinity for the second substance conjugated to the antibody. For example, the antibody may be conjugated to biotin and the antibody-biotin conjugate detected using labeled avidin or streptavidin. Similarly, the antibody may be conjugated to a hapten and the antibody-hapten conjugate detected using labeled anti-hapten antibody. These and other methods of labeling antibodies and assay conjugates are well known to those skilled in the art.

Further, when administered as pharmaceutical compositions to a wound or used to coat medical devices or polymeric biomaterials in vitro and in vivo, the antibodies of the present invention may be useful in those cases where there is a previous bacterial infection because of the ability of this antibody to further restrict and inhibit binding of Gram-positive bacteria to binding proteins such as fibrinogen or fibrin and thus limit the extent and spread of the infection. In addition, the antibody may be modified as necessary so that, in certain instances, it is less immunogenic in the patient to whom it is administered. For example, if the patient is a human, the antibody may be “humanized” by transplanting the complimentarity determining regions of the hybridoma-derived antibody into a human monoclonal antibody as described, e.g., by Jones et al., Nature 321:522-525 (1986) or Tempest et al. Biotechnology 9:266-273 (1991) or “veneered” by changing the surface exposed murine framework residues in the immunoglobulin variable regions to mimic a homologous human framework counterpart as described, e.g., by Padlan, Molecular lmm. 28:489-498 (1991), or European Patent application 519,596, these references incorporated herein by reference. Even further, when so desired, the monoclonal antibodies of the present invention may be administered in conjunction with a suitable antibiotic to further enhance the ability of the present compositions to fight bacterial infections.

Medical devices or polymeric biomaterials to be coated with the antibodies, proteins and active fragments described herein include, but are not limited to, staples, sutures, replacement heart valves, cardiac assist devices, hard and soft contact lenses, intraocular lens implants (anterior chamber or posterior chamber), other implants such as corneal inlays, kerato-prostheses, vascular stents, epikeratophalia devices, glaucoma shunts, retinal staples, scleral buckles, dental prostheses, thyroplastic devices, laryngoplastic devices, vascular grafts, soft and hard tissue prostheses including, but not limited to, pumps, electrical devices including stimulators and recorders, auditory prostheses, pacemakers, artificial larynx, dental implants, mammary implants, penile implants, cranio/facial tendons, artificial joints, tendons, ligaments, menisci, and disks, artificial bones, artificial organs including artificial pancreas, artificial hearts, artificial limbs, and heart valves; stents, wires, guide wires, intravenous and central venous catheters, laser and balloon angioplasty devices, vascular and heart devices (tubes, catheters, balloons), ventricular assists, blood dialysis components, blood oxygenators, urethral/ureteral/urinary devices (Foley catheters, stents, tubes and balloons), airway catheters (endotracheal and tracheostomy tubes and cuffs), enteral feeding tubes (including nasogastric, intragastric and jejunal tubes), wound drainage tubes, tubes used to drain the body cavities such as the pleural, peritoneal, cranial, and pericardial cavities, blood bags, test tubes, blood collection tubes, vacutainers, syringes, needles, pipettes, pipette tips, and blood tubing.

It will be understood by those skilled in the art that the term “coated” or “coating”, as used herein, means to apply the antibody or active fragment, or pharmaceutical composition derived therefrom, to a surface of the device, preferably an outer surface that would be exposed to a gram-positive bacterial infection. The surface of the device need not be entirely covered by the protein, antibody or active fragment.

In another embodiment of the invention, the antibodies may also be used as a passive vaccine which will be useful in providing suitable antibodies to treat or prevent a gram-positive bacterial infection. As would be recognized by one skilled in this art, such a vaccine may be packaged for administration in a number of suitable ways, such as by parenteral (i.e., intramuscular, intradermal or subcutaneous) administration or nasopharyngeal (i.e., intranasal) administration. One such mode is where the vaccine is injected intramuscularly, e.g., into the deltoid muscle. However, the particular mode of administration will depend on the nature of the bacterial infection to be dealt with and the condition of the patient. The vaccine is preferably combined with a pharmaceutically acceptable carrier to facilitate administration, and the carrier is usually water or a buffered saline, with or without a preservative. The vaccine may be lyophilized for resuspension at the time of administration or in solution.

The preferred dose for administration of an antibody composition in accordance with the present invention is that amount which will be effective in preventing or treating a gram-positive bacterial infection, and one would readily recognize that this amount will vary greatly depending on the nature of the infection and the condition of a patient. As indicated above, an “effective amount” of antibody or pharmaceutical agent to be used in accordance with the invention is intended to mean a nontoxic but sufficient amount of the agent, such that the desired prophylactic or therapeutic effect is produced. As pointed out below, the exact amount of the antibody or a particular agent that is required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular carrier or adjuvant being used and its mode of administration, and the like. Accordingly, the “effective amount” of any particular antibody composition will vary based on the particular circumstances, and an appropriate effective amount may be determined in each case of application by one of ordinary skill in the art using only routine experimentation. The dose should be adjusted to suit the individual to whom the composition is administered and will vary with age, weight and metabolism of the individual. The compositions may additionally contain stabilizers or pharmaceutically acceptable preservatives, such as thimerosal (ethyl(2-mercaptobenzoate-S)mercury sodium salt) (Sigma Chemical Company, St. Louis, Mo.).

When used with suitable labels or other appropriate detectable biomolecule or chemicals, the monoclonal antibodies described herein are useful for purposes such as in vivo and in vitro diagnosis of gram-positive bacterial infections or detection of gram-positive bacteria. Laboratory research may also be facilitated through use of such antibodies. Various types of labels and methods of conjugating the labels to the antibodies of the invention are well known to those skilled in the art, such as the ones set forth below.

For example, the antibody can be conjugated (directly or via chelation) to a radiolabel such as, but not restricted to, ³²p, ³H, ¹⁴C, ³⁵S, ¹²⁵I, or ¹³¹I. Detection of a label can be by methods such as scintillation counting, gamma ray spectrometry or autoradiography. Bioluminescent labels, such as derivatives of firefly luciferin, are also useful. The bioluminescent substance is covalently bound to the protein by conventional methods, and the labeled protein is detected when an enzyme, such as luciferase, catalyzes a reaction with ATP causing the bioluminescent molecule to emit photons of light. Fluorogens may also be used to label proteins. Examples of fluorogens include fluorescein and derivatives, phycoerythrin, allo-phycocyanin, phycocyanin, rhodamine, and Texas Red. The fluorogens are generally detected by a fluorescence detector.

The location of a ligand in cells can be determined by labeling an antibody as described above and detecting the label in accordance with methods well known to those skilled in the art, such as immunofluorescence microscopy using procedures such as those described by Warren and Nelson (Mol. Cell. Biol., 7: 1326-1337, 1987).

As indicated above, the antibodies of the present invention, or active portions or fragments thereof, are particularly useful for fighting or preventing bacteria infection in patients or on in-dwelling medical devices to make them safer for use. In short, the antibodies of the present invention are thus extremely useful in treating or preventing Gram-positive infections in human and animal patients and in medical or other in-dwelling devices.

In accordance with the invention, a diagnostic kit is also provided which utilizes an antibody of the invention as set forth above, and in one typical example, this kit may comprise an antibody of the invention which can recognize a conserved peptide region as set forth above or a protein containing said region, means for introducing the antibody to a sample suspected of containing gram-positive bacteria, and means for identifying gram-positive bacteria that are recognized by said antibody.

In accordance with the present invention, the peptides and proteins as described above may also be utilized in the development of vaccines for immunization against Gram-positive infections, and thus a method of eliciting an immune response in a human or animal is also provided wherein an immunogenic amount of a peptide or protein in accordance with the invention is administered to a human or animal. In the preferred embodiment, vaccines in accordance with the invention are prepared using methods that are conventionally used to prepare vaccines, and the preferred vaccine comprises an immunogenic amount of the peptides or proteins as described above along with a pharmaceutically acceptable vehicle, carrier or excipient. As would be recognized by one of ordinary skill in the art, these vaccines may be packaged for administration in a number of suitable ways, such as by parenteral (i.e., intramuscular, intradermal or subcutaneous) administration or nasopharyngeal (i.e., intranasal) administration. One such mode is where the vaccine is injected intramuscularly, e.g., into the deltoid muscle, however, the particular mode of administration will depend on the nature of the bacterial infection to be dealt with and the condition of the patient. The vaccine is preferably combined with a pharmaceutically acceptable vehicle, carrier or excipient in order to facilitate administration, and said carrier or other materials is usually water or a buffered saline, with or without a preservative. The vaccine may be lyophilized for resuspension at the time of administration or in solution.

The present invention thus provides for the identification and isolation of proteins having the signature conserved regions as set forth above, as well as the vaccines, antibodies and other forms of the invention as set forth above, and the invention will be particularly useful in developing and administering treatment regimens which can be used to fight or prevent infections caused by Gram-positive bacteria.

The following example is provided which exemplifies aspects of the preferred embodiments of the present invention. However, it will be appreciated by those of skill in the art that the techniques disclosed in the example which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. Moreover, those of skill in the art will also appreciate that in light of the present specification, many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention

EXAMPLE

Identification and Isolation of Conserved Sequences and Proteins Containing them

Gram-positive bacteria have a group of surface-located proteins that contain a unique sequence motif near the carboxyl termini. The motif consists of amino acid residues LPXTG (X being any amino acids) that is necessary for anchoring the protein to the bacterial cell wall by a transamidase called sortase. These bacterial surface proteins are thought to be important during the infection processes since they may mediate bacterial attachment to host tissues, and/or interact with the host immune system. They are potential candidates for active and/or passive immunization, as well as targets for new types of antibiotics. In Staphylococcus aureus, several of these proteins have been well characterized and were found to bind extracellular matrix proteins such as collagen, fibronectin, fibrinogen, as well as immunoglobulin G. The collagen and fibronectin binding proteins were shown to contribute to the virulence of S. aureus in animal models. In addition, immunization of mice with the collagen binding protein provided protection from septic death due to S. aureus, indicating that it may be used as a vaccine. LPXTG containing proteins that bind host proteins were also found in other gram-positive organisms such as Enterococcus faecalis and streptococci.

In this study we devised an algorithm for mining publicly available genome sequences of Gram-positive bacteria for LPXTG containing genes. We chose the genomes of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus Faecalis, Streptococcus pyogenes, Streptococcus pneumoniae, and Streptococcus mutans, all of which are important human pathogens. The genomes of four S. aureus strains were publicly available at the time of the analysis and were all included in the data mining process. The S. aureus genome sequences were obtained from the websites of The Institute for Genomic Research (TIGR) (strain COL), The Sanger Center (a methicillin resistant strain and a methicillin sensitive strain), and University of Oklahoma's Advanced Center for Genome Technology (OU-ACGT) (strain 8325). The genome sequences of E. faecalis (strain V583), S. epidermidis (strain RP62A) and S. pneumoniae Type 4 were obtained from TIGR, and the sequences of S. mutans and S. pyogenes (group A) were from OU-ACGT. Data mining was performed using a combination of software developed by us, Glimmer2 from TIGR and stand-alone BLAST from the National Center for Biotechnology Information. The system was set up on a Silicon Graphics machine running IRIX6.5. The algorithm consists the following steps: 1) process each sequence file which usually contains multiple contigs into individual files each of which consists one contig, 2) predict genes, 3) add unique identification tag to each predicted gene so that genes from different organisms can be put into one single database, 4) extract genes from each genome, 5) translate each gene into amino acid sequence, 6) form a blast searchable database of the protein sequences, and 7) blast search the database to find proteins that contain the LPXTG motif.

After LPXTG containing proteins were identified, they were collected into a subset and used to establish a separate blast searchable database. Each protein in this subset was blasted against each other as well as to the large protein database to identify LPXTG-containing proteins that are conserved among these organisms. Two groups were found. Members in each group exhibited substantial overall sequence homology with each other (see Tables 1 and 2 above). In addition, after multiple sequence alignment, there are stretches of completely identical sequences in each group (see FIGS. 1 and 2). Homology search with known genes indicated that the first group belongs to a family of cell division proteins, while the second group belongs to a family of amino acid transporters. However, none of the proteins in the two groups has been described for the organisms that we analyzed, and therefore they are novel for these bacteria.

Each protein in the two groups was examined for the presence of signal peptide through the SignalP mail server at Center for Biological Sequence Analysis, the Technical University of Denmark. Each was predicted to contain a signal peptide at the proper position, indicating that these are most likely surface proteins. Cell division proteins and amino acid transporters are important proteins for bacteria survival in vitro and in vivo. The fact that these proteins exhibit such high-level sequence conservation among the organisms suggests that they perform conserved functions. We envision that similar surface proteins are present in other Gram-positive bacteria. In fact we have identified 3 novel peptide sequences from the conserved proteins. The peptide sequences were selected from 3 regions in a Staphylococcus aureus protein that belongs to one ABC transporter group. Each region is highly conserved among the 6 Gram-positive bacteria examined (Enterococcus faecalis, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus mutans, Streptococcus pneumoniae, and Staphylococcus aureus).

Also, in order to increase the chance that the sequences will be exposed on the surface, we limited the selection of the sequences to hydrophilic regions using the method of Kyte and Doolittle. The sequences are listed below:

SA-1: ALKTG KIDII ISGMT STPER KK (SEQ ID NO:14)

SA-2: VEGAVVEKPVAEAYL KQN (SEQ ID NO:15)

SA-3: EYAGV DIDLA KKIAK (SEQ ID NO:16)

The peptides were synthesized in an Advanced Chem Tech 396 multiple peptide synthesizer, using Fmoc chemistry and activation with HBTU. After cleavage from the resin, peptides were purified by reverse-phase chromatography on a Waters Delta-Pak C18 column, eluted-with gradient of acetonitrile in 0.1% trifluoroacetic acid/water. The purity of the peptides was further confirmed by mass spectrometry analysis.

The peptide-KLH conjugation with EDC: The carrier protein KLH and the peptides (1:1 by weight) were coupled using EDC (Pierce) for 2 hours at room temperature. The reaction mixture is subjected to a desalting column pre-equilibrated with the purification buffer (0.083 M sodium phosphate, 0.9 M NaCl, pH 7.2). The conjugated peptides were eluted with the purification buffer and 0.5 ml fractions were collected. Each fraction was measured for its absorbance at 280 nm and the fractions containing the conjugate were pooled.

The use of the conserved conjugated peptides and polypeptides: The principle, methods and applications described above for the three conjugated peptides are applicable and will be applied to proteins in the second group of highly homologous surface proteins. This evidenced that: 1) antibodies raised against these proteins will be able to recognize a wide range of Gram-positive bacteria and may be used as a basis for a broad spectrum passive immunization protocol; 2) protective, therapeutic, or diagnostic antibodies raised against these proteins could recognize conserved epitopes present on different species of Gram-positive bacteria; 3) a single mAb recognizing the conserved peptides could be used to protect against all Gram-positive bacterial infections; 4) these proteins may be used as a basis for a broad spectrum vaccine; and 5) these proteins may be used as novel targets for designing new types of antimicrobial agents.

16 1 400 PRT Staphylococcus aureus 1 Met Asn Tyr Ser Ser Arg Gln Gln Pro Asp Lys His Trp Leu Arg Lys 1 5 10 15 Val Asp Trp Val Leu Val Ala Thr Ile Ala Val Leu Ala Ile Phe Ser 20 25 30 Val Leu Leu Ile Asn Ser Ala Met Gly Gly Gly Gln Tyr Ser Ala Asn 35 40 45 Phe Gly Ile Arg Gln Ile Phe Tyr Tyr Ile Leu Gly Ala Ile Phe Ala 50 55 60 Gly Ile Ile Met Phe Ile Ser Pro Lys Lys Ile Lys His Tyr Thr Tyr 65 70 75 80 Leu Leu Tyr Phe Leu Ile Cys Leu Leu Leu Ile Gly Leu Leu Val Ile 85 90 95 Pro Glu Ser Pro Ile Thr Pro Ile Ile Asn Gly Ala Lys Ser Trp Tyr 100 105 110 Thr Phe Gly Pro Ile Ser Ile Gln Pro Ser Glu Phe Met Lys Ile Ile 115 120 125 Leu Ile Leu Ala Leu Ala Arg Val Val Ser Arg His Asn Gln Phe Thr 130 135 140 Phe Asn Lys Ser Phe Gln Ser Asp Leu Leu Leu Phe Phe Lys Ile Ile 145 150 155 160 Gly Val Ser Leu Val Pro Ser Ile Leu Ile Leu Leu Gln Asn Asp Leu 165 170 175 Gly Thr Thr Leu Val Leu Ala Ala Ile Ile Ala Gly Val Met Leu Val 180 185 190 Ser Gly Ile Thr Trp Arg Ile Leu Ala Pro Ile Phe Ile Thr Gly Ile 195 200 205 Val Gly Ala Met Thr Val Ile Leu Gly Ile Leu Tyr Ala Pro Ala Leu 210 215 220 Ile Glu Asn Leu Leu Gly Val Gln Leu Tyr Gln Met Gly Arg Ile Asn 225 230 235 240 Ser Trp Leu Asp Pro Tyr Thr Tyr Ser Ser Gly Asp Gly Tyr His Leu 245 250 255 Thr Glu Ser Leu Lys Ala Ile Gly Ser Gly Gln Leu Leu Gly Lys Gly 260 265 270 Tyr Asn His Gly Glu Val Tyr Ile Pro Glu Asn His Thr Asp Phe Ile 275 280 285 Phe Ser Val Ile Gly Glu Glu Leu Gly Phe Ile Gly Ser Val Ile Leu 290 295 300 Ile Leu Ile Phe Leu Phe Leu Ile Phe His Leu Ile Arg Leu Ala Ala 305 310 315 320 Lys Ile Glu Asp Gln Phe Asn Lys Ile Phe Ile Val Gly Phe Val Thr 325 330 335 Leu Leu Val Phe His Ile Leu Gln Asn Ile Gly Met Thr Ile Gln Leu 340 345 350 Leu Pro Ile Thr Gly Ile Pro Leu Pro Phe Ile Ser Tyr Gly Gly Ser 355 360 365 Ala Leu Trp Ser Met Met Thr Gly Ile Gly Ile Val Leu Ser Ile Tyr 370 375 380 Tyr His Glu Pro Lys Arg Tyr Val Asp Leu Tyr His Pro Lys Ser Asn 385 390 395 400 2 403 PRT Staphylococcus epidermidis 2 Met Asn Tyr Ser Ser Arg Gln Gln Pro Lys Arg Asn Trp Leu Arg Lys 1 5 10 15 Val Asp Trp Ile Leu Val Leu Val Ile Ser Leu Leu Ala Leu Thr Ser 20 25 30 Val Ile Leu Ile Ser Ser Ala Met Gly Gly Gly Gln Tyr Ser Ala Asn 35 40 45 Phe Ser Ile Arg Gln Ile Ile Tyr Tyr Ile Phe Gly Ala Ile Ile Ala 50 55 60 Phe Leu Ile Met Ile Ile Ser Pro Lys Lys Ile Lys Asn Asn Thr Tyr 65 70 75 80 Ile Leu Tyr Ser Ile Phe Cys Val Leu Leu Ile Gly Leu Leu Ile Leu 85 90 95 Pro Glu Thr Ser Ile Thr Pro Ile Ile Asn Gly Ala Lys Ser Trp Tyr 100 105 110 Ser Phe Gly Pro Ile Ser Ile Gln Pro Ser Glu Phe Met Lys Ile Ile 115 120 125 Leu Ile Leu Ala Leu Ala Lys Thr Ile Ser Lys His Asn Gln Phe Thr 130 135 140 Phe Asn Lys Ser Phe Gln Ser Asp Leu Met Leu Phe Phe Lys Ile Leu 145 150 155 160 Gly Val Ser Ile Ile Pro Met Ala Leu Ile Leu Leu Gln Asn Asp Leu 165 170 175 Gly Thr Thr Leu Val Leu Cys Ala Ile Ile Ala Gly Val Met Leu Val 180 185 190 Ser Gly Ile Thr Trp Arg Ile Leu Ala Pro Leu Phe Ile Val Ala Phe 195 200 205 Val Ser Gly Ser Ser Ile Ile Leu Ala Ile Ile Tyr Lys Pro Ser Leu 210 215 220 Ile Glu Asn Leu Leu Gly Ile Lys Met Tyr Gln Met Gly Arg Ile Asn 225 230 235 240 Ser Trp Leu Asp Pro Tyr Ser Tyr Ser Ser Gly Asp Gly Tyr His Leu 245 250 255 Thr Glu Ser Leu Lys Ala Ile Gly Ser Gly Gln Leu Leu Gly Lys Gly 260 265 270 Tyr Asn His Gly Glu Val Tyr Ile Pro Glu Asn His Thr Asp Phe Ile 275 280 285 Phe Ser Val Ile Gly Glu Glu Met Gly Phe Ile Gly Ser Val Leu Leu 290 295 300 Ile Leu Leu Phe Leu Phe Leu Ile Phe His Leu Ile Arg Leu Ala Ser 305 310 315 320 Lys Ile Asp Ser Gln Phe Asn Lys Val Phe Ile Ile Gly Tyr Val Ser 325 330 335 Leu Ile Val Phe His Val Leu Gln Asn Ile Gly Met Thr Val Gln Leu 340 345 350 Leu Pro Ile Thr Gly Ile Pro Leu Pro Phe Ile Ser Tyr Gly Gly Ser 355 360 365 Ser Leu Trp Ser Leu Met Thr Gly Ile Gly Val Val Leu Ser Ile Tyr 370 375 380 Tyr His Glu Pro Gln Arg Tyr Glu Ile Thr Thr Leu Ser Lys Lys Ser 385 390 395 400 Asn Thr Ile 3 408 PRT Streptococcus mutans 3 Met Ala Ser Lys Lys Lys Pro Ile Asp Ser Arg Val Asp Tyr Ser Leu 1 5 10 15 Ile Leu Pro Val Phe Phe Leu Val Leu Ile Gly Leu Phe Ser Val Tyr 20 25 30 Thr Ala Thr Ile His Asp Tyr Pro Ser Lys Ile Met Val Val Met Gly 35 40 45 Gln Gln Leu Ile Trp Leu Ile Met Gly Ala Ala Ile Ser Phe Val Val 50 55 60 Met Leu Phe Ser Thr Glu Phe Leu Trp Lys Ile Thr Pro Tyr Leu Tyr 65 70 75 80 Gly Leu Gly Leu Ile Leu Met Ile Phe Pro Leu Ile Phe Tyr Ser Pro 85 90 95 Glu Leu Val Ala Ser Thr Gly Ala Lys Asn Trp Val Ser Ile Gly Ser 100 105 110 Val Thr Leu Phe Gln Pro Ser Glu Phe Met Lys Ile Ser Tyr Ile Leu 115 120 125 Ile Leu Ala Arg Leu Thr Val Thr Phe Lys Gln Lys Tyr Lys Glu Lys 130 135 140 Asn Leu Gln Glu Asp Gly Lys Leu Leu Leu Trp Phe Ala Leu Leu Thr 145 150 155 160 Leu Pro Ile Met Ile Leu Leu Ala Leu Gln Lys Asp Leu Gly Thr Ala 165 170 175 Met Val Phe Met Ala Ile Leu Ala Gly Leu Val Leu Ile Ala Gly Ile 180 185 190 Ser Trp Gln Ile Ile Leu Pro Val Val Gly Ala Val Ala Leu Ile Val 195 200 205 Ala Leu Phe Met Val Val Phe Leu Ile Pro Gly Gly Lys Glu Phe Leu 210 215 220 Tyr His His Met Gly Val Asp Thr Tyr Gln Ile Asn Arg Leu Ser Ala 225 230 235 240 Trp Leu Asn Pro Phe Asp Tyr Ala Gly Ser Ile Ala Tyr Gln Gln Thr 245 250 255 Gln Gly Met Ile Ser Ile Gly Ser Gly Gly Leu Phe Gly Lys Gly Phe 260 265 270 Asn Ile Val Glu Leu Pro Val Pro Val Arg Glu Ser Asp Met Ile Phe 275 280 285 Thr Val Ile Ala Glu Asn Phe Gly Phe Ile Gly Gly Ser Ile Val Leu 290 295 300 Ala Leu Tyr Leu Ile Leu Ile Tyr Arg Met Leu Arg Val Thr Phe Ala 305 310 315 320 Ser Asn Asn Leu Phe Tyr Thr Tyr Ile Ser Thr Gly Phe Ile Met Met 325 330 335 Ile Leu Phe His Ile Phe Glu Asn Ile Gly Ala Ala Val Gly Ile Leu 340 345 350 Pro Leu Thr Gly Ile Pro Leu Pro Phe Ile Ser Gln Gly Gly Ser Ser 355 360 365 Leu Ile Ser Asn Leu Ile Gly Val Gly Leu Val Leu Ser Met Ser Tyr 370 375 380 Gln Asn Ser Leu Asn Gln Glu Lys Ala Thr Glu Arg Tyr Phe Ala His 385 390 395 400 Ile Lys Lys Glu Ser Leu Thr Ser 405 4 416 PRT Streptococcus pneumoniae 4 Leu Tyr Glu Ser Ile Arg Leu Val Tyr Met Lys Arg Ser Leu Asp Ser 1 5 10 15 Arg Val Asp Tyr Ser Leu Leu Leu Pro Val Phe Phe Leu Leu Val Ile 20 25 30 Gly Val Val Ala Ile Tyr Ile Ala Val Ser His Asp Tyr Pro Asn Asn 35 40 45 Ile Leu Pro Ile Leu Gly Gln Gln Val Ala Trp Ile Ala Leu Gly Leu 50 55 60 Val Ile Gly Phe Val Val Met Leu Phe Asn Thr Glu Phe Leu Trp Lys 65 70 75 80 Val Thr Pro Phe Leu Tyr Ile Leu Gly Leu Gly Leu Met Ile Leu Pro 85 90 95 Ile Val Phe Tyr Asn Pro Ser Leu Val Ala Ser Thr Gly Ala Lys Asn 100 105 110 Trp Val Ser Ile Asn Gly Ile Thr Leu Phe Gln Pro Ser Glu Phe Met 115 120 125 Lys Ile Ser Tyr Ile Leu Met Leu Ala Arg Val Ile Val Gln Phe Thr 130 135 140 Lys Lys His Lys Glu Trp Arg Arg Thr Val Pro Leu Asp Phe Leu Leu 145 150 155 160 Ile Phe Trp Met Ile Leu Phe Thr Ile Pro Val Leu Val Leu Leu Ala 165 170 175 Leu Gln Ser Asp Leu Gly Thr Ala Leu Val Phe Val Ala Ile Phe Ser 180 185 190 Gly Ile Val Leu Leu Ser Gly Val Ser Trp Lys Ile Ile Ile Pro Val 195 200 205 Phe Val Thr Ala Val Thr Gly Val Ala Gly Phe Leu Ala Ile Phe Ile 210 215 220 Ser Lys Asp Gly Arg Ala Phe Leu His Gln Ile Gly Met Pro Thr Tyr 225 230 235 240 Gln Ile Asn Arg Ile Leu Ala Trp Leu Asn Pro Phe Glu Phe Ala Gln 245 250 255 Thr Thr Thr Tyr Gln Gln Ala Gln Gly Gln Ile Ala Ile Gly Ser Gly 260 265 270 Gly Leu Phe Gly Gln Gly Phe Asn Ala Ser Asn Leu Leu Ile Pro Val 275 280 285 Arg Glu Ser Asp Met Ile Phe Thr Val Ile Ala Glu Asp Phe Gly Phe 290 295 300 Ile Gly Ser Val Leu Val Ile Ala Leu Tyr Leu Met Leu Ile Tyr Arg 305 310 315 320 Met Leu Lys Ile Thr Leu Lys Ser Asn Asn Gln Phe Tyr Thr Tyr Ile 325 330 335 Ser Thr Gly Leu Ile Met Met Leu Leu Phe His Ile Phe Glu Asn Ile 340 345 350 Gly Ala Val Thr Gly Leu Leu Pro Leu Thr Gly Ile Pro Leu Pro Phe 355 360 365 Ile Ser Gln Gly Gly Ser Ala Ile Ile Ser Asn Leu Ile Gly Val Gly 370 375 380 Leu Leu Leu Ser Met Ser Tyr Gln Thr Asn Leu Ala Glu Glu Lys Ser 385 390 395 400 Gly Lys Val Pro Phe Lys Arg Lys Lys Val Val Leu Lys Gln Ile Lys 405 410 415 5 395 PRT Enterococcus faecalis 5 Met Asn Arg Lys Glu Lys Thr Asn Leu Asp Ser Arg Ile Asp Tyr Gly 1 5 10 15 Val Ile Leu Pro Val Phe Leu Leu Ser Leu Ile Gly Met Leu Ser Leu 20 25 30 Tyr Val Ala Leu Tyr Asn Asp Pro Ser Lys Pro Lys Ile Gly Ser Leu 35 40 45 Leu Met Lys Gln Gly Leu Trp Tyr Leu Val Gly Gly Leu Ser Ile Val 50 55 60 Ile Ile Met His Phe Ser Ser Lys Leu Leu Trp Arg Leu Thr Pro Val 65 70 75 80 Phe Tyr Ala Leu Gly Leu Val Leu Met Gly Leu Leu Leu Lys Phe Tyr 85 90 95 Asp Pro Val Leu Ala Glu Gln Thr Gly Ser Lys Asn Trp Ile Arg Phe 100 105 110 Gly Gly Thr Thr Phe Gln Pro Ser Glu Leu Met Lys Ile Ala Phe Ile 115 120 125 Leu Met Leu Ala Tyr Ile Val Thr Met His Asn Val Lys Tyr Val Asp 130 135 140 Arg Thr Leu Lys Ser Asp Phe Trp Leu Ile Ala Lys Met Leu Leu Val 145 150 155 160 Ala Ile Pro Val Ile Val Leu Val Leu Leu Gln Lys Asp Phe Gly Thr 165 170 175 Met Leu Val Phe Leu Ala Ile Phe Gly Gly Val Phe Leu Met Ser Gly 180 185 190 Ile Thr Trp Lys Ile Ile Val Pro Val Phe Ile Leu Ala Ala Leu Val 195 200 205 Gly Ala Gly Thr Ile Tyr Leu Ile Thr Thr Glu Thr Gly Arg Asp Leu 210 215 220 Leu Ser Lys Leu Gly Val Glu Ala Tyr Lys Phe Asp Arg Ile Asp Leu 225 230 235 240 Trp Leu Asn Pro Phe His Thr Asp Pro Asp Arg Ser Phe Gln Pro Ala 245 250 255 Leu Ala Leu Thr Ala Ile Gly Ser Gly Gly Leu Phe Gly Lys Gly Phe 260 265 270 Asn Val Ser Asp Val Tyr Val Pro Val Arg Glu Ser Asp Met Ile Phe 275 280 285 Thr Val Val Gly Glu Asn Phe Gly Phe Ile Gly Gly Cys Phe Ile Ile 290 295 300 Leu Leu Tyr Phe Ile Leu Ile Tyr Arg Met Ile Arg Val Cys Phe Asp 305 310 315 320 Thr Asn Asn Glu Phe Tyr Ala Tyr Ile Ala Thr Gly Ile Ile Met Met 325 330 335 Ile Leu Phe His Val Phe Glu Asn Ile Gly Ala Asn Ile Gly Leu Leu 340 345 350 Pro Leu Thr Gly Ile Pro Leu Pro Phe Ile Ser Gln Gly Gly Ser Ser 355 360 365 Ile Leu Gly Asn Met Ile Gly Val Gly Leu Ile Met Ser Met Arg Tyr 370 375 380 Gln Gln Glu Thr Val Arg Thr Arg Ser Gly Arg 385 390 395 6 434 PRT Streptococcus pyogenes 6 Met Ile Ile Ser Arg Ser Arg Gly Lys Thr Met Lys Ile Asp Lys Arg 1 5 10 15 His Leu Leu Asn Tyr Ser Ile Leu Leu Pro Tyr Leu Ile Leu Ser Val 20 25 30 Ile Gly Leu Ile Met Val Tyr Ser Thr Thr Ser Val Ser Leu Ile Gln 35 40 45 Ala His Ala Asn Pro Phe Lys Ser Val Ile Asn Gln Gly Val Phe Trp 50 55 60 Ile Ile Ser Leu Val Ala Ile Thr Phe Ile Tyr Lys Leu Lys Leu Asn 65 70 75 80 Phe Leu Thr Asn Thr Arg Val Leu Thr Val Val Met Leu Gly Glu Ala 85 90 95 Phe Leu Leu Ile Ile Ala Arg Phe Phe Thr Thr Ala Ile Lys Gly Ala 100 105 110 His Gly Trp Ile Val Ile Gly Pro Val Ser Phe Gln Pro Ala Glu Tyr 115 120 125 Leu Lys Ile Ile Met Val Trp Tyr Leu Ala Leu Thr Phe Ala Lys Ile 130 135 140 Gln Lys Asn Ile Ser Leu Tyr Asp Tyr Gln Ala Leu Thr Arg Arg Lys 145 150 155 160 Trp Trp Pro Thr Gln Trp Asn Asp Leu Arg Asp Trp Arg Val Tyr Ser 165 170 175 Leu Leu Met Val Leu Leu Val Ala Ala Gln Pro Asp Leu Gly Asn Ala 180 185 190 Ser Ile Ile Val Leu Thr Ala Ile Ile Met Phe Ser Ile Ser Gly Ile 195 200 205 Gly Tyr Arg Trp Phe Ser Ala Ile Leu Val Met Ile Thr Gly Leu Ser 210 215 220 Thr Val Phe Leu Gly Thr Ile Ala Val Ile Gly Val Glu Arg Val Ala 225 230 235 240 Lys Ile Pro Val Phe Gly Tyr Val Ala Lys Arg Phe Ser Ala Phe Phe 245 250 255 Asn Pro Phe His Asp Leu Thr Asp Ser Gly His Gln Leu Ala Asn Ser 260 265 270 Tyr Tyr Ala Met Ser Asn Gly Gly Trp Phe Gly Gln Gly Leu Gly Asn 275 280 285 Ser Ile Glu Lys Arg Gly Tyr Leu Pro Glu Ala Gln Thr Asp Phe Val 290 295 300 Phe Ser Val Val Ile Glu Glu Leu Gly Leu Ile Gly Ala Gly Phe Ile 305 310 315 320 Leu Ala Leu Val Phe Phe Leu Ile Leu Arg Ile Met Asn Val Gly Ile 325 330 335 Lys Ala Lys Asn Pro Phe Asn Ala Met Met Ala Leu Gly Val Gly Gly 340 345 350 Met Met Leu Met Gln Val Phe Val Asn Ile Gly Gly Ile Ser Gly Leu 355 360 365 Ile Pro Ser Thr Gly Val Thr Phe Pro Phe Leu Ser Gln Gly Gly Asn 370 375 380 Ser Leu Leu Val Leu Ser Val Ala Val Gly Phe Val Leu Asn Ile Asp 385 390 395 400 Ala Ser Glu Lys Arg Asp Asp Ile Phe Lys Glu Ala Glu Leu Ser Tyr 405 410 415 Arg Lys Asp Thr Arg Lys Glu Asn Ser Lys Val Val Asn Ile Lys Gln 420 425 430 Phe Gln 7 528 PRT Streptococcus pyogenes 7 Leu Lys Gln Glu Thr Tyr Met Lys Lys Leu Ile Leu Ser Cys Leu Val 1 5 10 15 Ala Leu Ala Leu Leu Phe Gly Gly Met Ser Arg Ala Gln Ala Asn Gln 20 25 30 Tyr Leu Arg Val Gly Met Glu Ala Ala Tyr Ala Pro Phe Asn Trp Thr 35 40 45 Gln Asp Asp Ala Ser Asn Gly Ala Val Pro Ile Glu Gly Thr Ser Gln 50 55 60 Tyr Ala Asn Gly Tyr Asp Val Gln Val Ala Lys Lys Val Ala Lys Ala 65 70 75 80 Met Asn Lys Glu Leu Leu Val Val Lys Thr Ser Trp Thr Gly Leu Ile 85 90 95 Pro Ala Leu Thr Ser Gly Lys Ile Asp Met Ile Ala Ala Gly Met Ser 100 105 110 Pro Thr Lys Glu Arg Arg Asn Glu Ile Ser Phe Ser Asn Ser Ser Tyr 115 120 125 Thr Ser Gln Pro Val Leu Val Val Thr Ala Asn Gly Lys Tyr Ala Asp 130 135 140 Ala Thr Ser Leu Lys Asp Phe Ser Gly Ala Lys Val Thr Ala Gln Gln 145 150 155 160 Gly Val Trp His Val Asn Leu Leu Thr Gln Leu Lys Gly Ala Lys Leu 165 170 175 Gln Thr Pro Met Gly Asp Phe Ser Gln Met Arg Gln Ala Leu Thr Ser 180 185 190 Gly Val Ile Asp Ala Tyr Ile Ser Glu Arg Pro Glu Ala Met Thr Ala 195 200 205 Glu Ala Ala Asp Ser Arg Leu Lys Met Ile Thr Leu Lys Lys Gly Phe 210 215 220 Ala Val Ala Glu Ser Asp Ala Ala Ile Ala Val Gly Met Lys Lys Asn 225 230 235 240 Asp Asp Arg Met Ala Thr Val Asn Gln Val Leu Glu Gly Phe Ser Gln 245 250 255 Thr Asp Arg Met Ala Leu Met Asp Asp Met Val Thr Lys Gln Pro Val 260 265 270 Glu Lys Lys Ala Glu Asp Ala Lys Ala Ser Phe Leu Gly Gln Met Trp 275 280 285 Ala Ile Phe Lys Gly Asn Trp Lys Gln Phe Leu Arg Gly Thr Gly Met 290 295 300 Thr Leu Leu Ile Ser Met Val Gly Thr Ile Thr Gly Leu Phe Ile Gly 305 310 315 320 Leu Leu Ile Gly Ile Phe Arg Thr Ala Pro Lys Ala Lys His Lys Val 325 330 335 Ala Ala Leu Gly Gln Lys Leu Phe Gly Trp Leu Leu Thr Ile Tyr Ile 340 345 350 Glu Ile Phe Arg Gly Thr Pro Met Ile Val Gln Ser Met Val Ile Tyr 355 360 365 Tyr Gly Thr Ala Gln Ala Phe Gly Ile Ser Ile Asp Arg Thr Leu Ala 370 375 380 Ala Ile Phe Ile Val Ser Ile Asn Thr Gly Ala Tyr Met Ser Glu Ile 385 390 395 400 Val Arg Gly Gly Ile Phe Ala Val Asp Lys Gly Gln Phe Lys Ala Ala 405 410 415 Thr Ala Leu Gly Phe Thr His Gly Gln Thr Met Arg Lys Ile Val Leu 420 425 430 Pro Gln Val Val Arg Asn Ile Leu Pro Ala Thr Gly Asn Glu Phe Val 435 440 445 Ile Asn Ile Lys Asp Thr Ser Val Leu Asn Val Ile Ser Val Val Glu 450 455 460 Leu Tyr Phe Ser Gly Asn Thr Val Ala Thr Gln Thr Tyr Gln Tyr Phe 465 470 475 480 Gln Thr Phe Thr Ile Ile Ala Ile Ile Tyr Phe Val Leu Thr Phe Thr 485 490 495 Val Thr Arg Ile Leu Arg Tyr Ile Glu Arg Arg Phe Asp Ala Asp Thr 500 505 510 Tyr Thr Thr Gly Ala Asn Gln Met Gln Ile Ala Glu Val Ser Asn Val 515 520 525 8 521 PRT Streptococcus pneumoniae 8 Met Arg Lys Ile Tyr Leu Ser Ile Phe Thr Ser Leu Leu Leu Met Leu 1 5 10 15 Gly Leu Val Asn Val Ala Gln Ala Asp Glu Tyr Leu Arg Ile Gly Met 20 25 30 Glu Ala Ala Tyr Ala Pro Phe Asn Trp Thr Gln Asp Asp Asp Ser Asn 35 40 45 Gly Ala Val Lys Ile Asp Gly Thr Asn Gln Tyr Ala Asn Gly Tyr Asp 50 55 60 Val Gln Ile Ala Lys Lys Ile Ala Lys Asp Leu Gly Lys Glu Pro Leu 65 70 75 80 Val Val Lys Thr Lys Trp Glu Gly Leu Val Pro Ala Leu Thr Ser Gly 85 90 95 Lys Ile Asp Met Ile Ile Ala Gly Met Ser Pro Thr Ala Glu Arg Lys 100 105 110 Gln Glu Ile Ala Phe Ser Ser Ser Tyr Tyr Thr Ser Glu Pro Val Leu 115 120 125 Leu Val Lys Lys Asp Ser Ala Tyr Ala Ser Ala Lys Ser Leu Asp Asp 130 135 140 Phe Asn Gly Ala Lys Ile Thr Ser Gln Gln Gly Val Tyr Leu Tyr Asn 145 150 155 160 Leu Ile Ala Gln Ile Pro Gly Ala Lys Lys Glu Thr Ala Met Gly Asp 165 170 175 Phe Ala Gln Met Arg Gln Ala Leu Glu Ala Gly Val Ile Asp Ala Tyr 180 185 190 Val Ser Glu Arg Pro Glu Ala Leu Thr Ala Glu Ala Ala Asn Ser Lys 195 200 205 Phe Lys Met Ile Gln Val Glu Pro Gly Phe Lys Thr Gly Glu Glu Asp 210 215 220 Thr Ala Ile Ala Ile Gly Leu Arg Lys Asn Asp Asn Arg Ile Ser Gln 225 230 235 240 Ile Asn Ala Ser Ile Glu Thr Ile Ser Lys Asp Asp Gln Val Ala Leu 245 250 255 Met Asp Arg Met Ile Lys Glu Gln Pro Ala Glu Ala Thr Thr Thr Glu 260 265 270 Glu Thr Ser Ser Ser Phe Phe Ser Gln Val Ala Lys Ile Leu Ser Glu 275 280 285 Asn Trp Gln Gln Leu Leu Arg Gly Ala Gly Ile Thr Leu Leu Ile Ser 290 295 300 Ile Val Gly Thr Ile Ile Gly Leu Ile Ile Gly Leu Ala Ile Gly Val 305 310 315 320 Phe Arg Thr Ala Pro Leu Ser Glu Asn Lys Val Ile Tyr Gly Leu Gln 325 330 335 Lys Leu Val Gly Trp Val Leu Asn Val Tyr Ile Glu Ile Phe Arg Gly 340 345 350 Thr Pro Met Ile Val Gln Ser Met Val Ile Tyr Tyr Gly Thr Ala Gln 355 360 365 Ala Phe Gly Ile Asn Leu Asp Arg Thr Leu Ala Ala Ile Phe Ile Val 370 375 380 Ser Ile Asn Thr Gly Ala Tyr Met Thr Glu Ile Val Arg Gly Gly Ile 385 390 395 400 Leu Ala Val Asp Lys Gly Gln Phe Glu Ala Ala Thr Ala Leu Gly Met 405 410 415 Thr His Asn Gln Thr Met Arg Lys Ile Val Leu Pro Gln Val Val Arg 420 425 430 Asn Ile Leu Pro Ala Thr Gly Asn Glu Phe Val Ile Asn Ile Lys Asp 435 440 445 Thr Ser Val Leu Asn Val Ile Ser Val Val Glu Leu Tyr Phe Ser Gly 450 455 460 Asn Thr Val Ala Thr Gln Thr Tyr Gln Tyr Phe Gln Thr Phe Thr Ile 465 470 475 480 Ile Ala Val Ile Tyr Phe Val Leu Thr Phe Thr Val Thr Arg Ile Leu 485 490 495 Arg Phe Ile Glu Arg Arg Met Asp Met Asp Thr Tyr Thr Thr Gly Ala 500 505 510 Asn Gln Met Gln Thr Glu Asp Leu Lys 515 520 9 517 PRT Streptococcus mutans 9 Met Lys Lys Thr Ile Leu Ser Cys Leu Ala Ala Leu Phe Met Leu Phe 1 5 10 15 Ile Gly Val Thr Asn Ala Gln Ala Asp Asn Tyr Leu Arg Val Gly Met 20 25 30 Glu Ala Ala Tyr Ala Pro Phe Asn Trp Thr Gln Asp Asn Ser Ser Asn 35 40 45 Gly Ala Val Pro Ile Glu Gly Thr Lys Gln Tyr Ala Asn Gly Tyr Asp 50 55 60 Val Gln Thr Ala Lys Lys Ile Ala Lys Thr Leu Gly Lys Lys Pro Leu 65 70 75 80 Ile Val Lys Thr Lys Trp Glu Gly Leu Val Pro Ala Leu Thr Ser Gly 85 90 95 Lys Ile Asp Leu Ile Ile Ala Gly Met Ser Pro Thr Lys Glu Arg Lys 100 105 110 Lys Glu Ile Ala Phe Ser Asn Ser Tyr Tyr Thr Ser Glu Pro Val Leu 115 120 125 Val Val Arg Lys Asp Ser Lys Tyr Ala Lys Ala Lys Asn Leu Asn Asp 130 135 140 Phe Ser Gly Ala Lys Val Thr Ser Gln Gln Gly Val Tyr Leu Tyr Asn 145 150 155 160 Leu Ile Asn Gln Ile Pro Lys Val Ser Arg Gln Thr Ala Met Gly Asp 165 170 175 Phe Ser Gln Met Arg Gln Ala Leu Ala Ser Asn Val Ile Asp Ala Tyr 180 185 190 Val Ser Glu Arg Pro Glu Ala Leu Ser Ser Thr Lys Ala Asn Ser Asn 195 200 205 Phe Lys Met Val Ser Leu Lys Asn Gly Phe Lys Val Ser Lys Ser Asp 210 215 220 Val Thr Ile Ala Val Gly Met Arg Lys Gly Asp Pro Arg Ile Glu Gln 225 230 235 240 Val Asn Ala Ala Leu Asp Gln Phe Pro Leu Lys Glu Gln Ile Ser Leu 245 250 255 Met Asp Lys Ile Ile Pro Met Gln Pro Ser Gln Asn Asn Ser Asp Gln 260 265 270 Lys Glu Ser Lys Ser Asn Phe Phe Asp Gln Val Ser Lys Ile Val Lys 275 280 285 Asn Asn Trp Lys Ala Leu Leu Arg Gly Thr Gly Val Thr Leu Leu Ile 290 295 300 Ser Ile Ile Gly Thr Ile Ala Gly Leu Ile Ile Gly Leu Leu Ile Gly 305 310 315 320 Val Tyr Arg Thr Ala Pro Lys Ala Ser Asn Leu Ile Leu Ala Trp Leu 325 330 335 Gln Lys Ile Phe Gly Trp Leu Leu Thr Val Tyr Ile Glu Val Phe Arg 340 345 350 Gly Thr Pro Met Ile Val Gln Ala Met Val Ile Tyr Tyr Gly Thr Ala 355 360 365 Gln Ala Phe Gly Val Ser Leu Asp Arg Thr Leu Ala Ala Ile Phe Ile 370 375 380 Val Ser Ile Asn Thr Gly Ala Tyr Met Ser Glu Ile Val Arg Gly Gly 385 390 395 400 Ile Phe Ala Val Asp Lys Gly Gln Phe Glu Ala Ala Thr Ala Leu Gly 405 410 415 Phe Thr His Arg Gln Thr Met Arg Lys Ile Val Leu Pro Gln Val Val 420 425 430 Arg Asn Ile Leu Pro Ala Thr Gly Asn Glu Phe Val Ile Asn Ile Lys 435 440 445 Asp Thr Ser Val Leu Asn Val Ile Ser Val Val Glu Leu Tyr Phe Ser 450 455 460 Gly Asn Thr Val Ala Thr Gln Thr Tyr Gln Tyr Phe Gln Thr Phe Phe 465 470 475 480 Ile Ile Ala Val Ile Tyr Phe Ile Leu Thr Phe Thr Val Thr Arg Ile 485 490 495 Leu Arg Leu Val Glu Arg Lys Met Asp Gln Asp Asn Tyr Thr Lys Ile 500 505 510 Glu Gly Glu Thr Asn 515 10 547 PRT Enterococcus faecalis 10 Leu Leu Ile Glu Lys Arg Gln Asn Asp Gln Ser Asp Lys Lys Phe Lys 1 5 10 15 Gly Glu Lys Lys Met Asn Lys Lys Val Phe Ser Phe Ser Leu Leu Leu 20 25 30 Val Thr Leu Phe Ser Leu Leu Gly Met Thr Thr Asn Ala Ser Ala Glu 35 40 45 Glu Asn Gly Glu Phe Arg Val Gly Met Glu Ala Gly Tyr Ala Pro Phe 50 55 60 Asn Trp Ser Gln Lys Asn Asp Ala His Gly Ala Val Pro Ile Gln Gly 65 70 75 80 Asn Ser Tyr Ala Gly Gly Tyr Asp Val Gln Ile Ser Lys Lys Ile Ala 85 90 95 Asp Gly Leu Gly Arg Lys Leu Val Ile Val Gln Thr Lys Trp Asp Gly 100 105 110 Leu Ala Pro Ala Leu Gln Ser Gly Lys Ile Asp Ala Ile Ile Ala Gly 115 120 125 Met Ser Pro Thr Ala Glu Arg Lys Lys Glu Ile Ala Phe Thr Asn Pro 130 135 140 Tyr Tyr Glu Ser Gln Phe Val Val Ile Val Lys Lys Asp Gly Lys Tyr 145 150 155 160 Ala Asn Ala Lys Ser Leu Lys Asp Leu Ala Asp Ala Lys Ile Thr Ala 165 170 175 Gln Leu Asn Thr Phe His Tyr Gly Leu Ile Asp Gln Ile Pro Asn Val 180 185 190 Asn Lys Gln Gln Ala Met Asp Asn Phe Ser Ala Met Arg Thr Ala Leu 195 200 205 Ala Ser Gly Met Ile Asp Gly Tyr Val Ser Glu Arg Pro Glu Gly Ile 210 215 220 Thr Ala Thr Ser Val Asn Lys Glu Leu Lys Met Leu Glu Phe Pro Lys 225 230 235 240 Glu Lys Gly Phe Asp Ala Ser Ala Glu Asp Ser Gln Val Ala Val Gly 245 250 255 Met Arg Lys Gly Asp Pro Asp Ile Glu Lys Val Asn Lys Ile Leu Ala 260 265 270 Gly Ile Ser Gln Asp Glu Arg Thr Lys Ile Met Asp Gln Ala Ile Lys 275 280 285 Asp Gln Pro Ala Ala Thr Asp Ser Asp Glu Gln Lys Thr Gly Leu Ile 290 295 300 Asn Asp Phe Lys Asn Ile Trp Asn Gln Tyr Gly Asp Met Phe Leu Arg 305 310 315 320 Gly Ala Gly Leu Thr Leu Phe Ile Ala Leu Ile Gly Thr Val Val Gly 325 330 335 Thr Thr Leu Gly Leu Leu Ile Gly Val Phe Arg Thr Ile Pro Asp Ser 340 345 350 Glu Asn Pro Val Ala Arg Phe Phe Gln Lys Leu Gly Asn Leu Ile Leu 355 360 365 Ser Ile Tyr Ile Glu Val Phe Arg Gly Thr Pro Met Met Val Gln Ala 370 375 380 Met Val Ile Phe Tyr Gly Leu Ala Leu Ala Phe Gly Ile Ser Leu Asp 385 390 395 400 Arg Thr Val Ala Ala Leu Phe Ile Val Ser Val Asn Thr Gly Ala Tyr 405 410 415 Met Ser Glu Ile Val Arg Gly Gly Ile Phe Ala Val Asp Lys Gly Gln 420 425 430 Phe Glu Ala Ala Gln Ala Ile Gly Met Thr His Gly Gln Thr Met Arg 435 440 445 Lys Val Val Ile Pro Gln Val Leu Arg Asn Ile Leu Pro Ala Thr Gly 450 455 460 Asn Glu Phe Val Ile Asn Ile Lys Asp Thr Ala Val Leu Ser Val Ile 465 470 475 480 Gly Val Ala Asp Leu Phe Phe Gln Gly Asn Ala Ala Ser Gly Ala Asn 485 490 495 Phe Gln Phe Phe Gln Thr Phe Thr Ile Val Gly Ile Met Tyr Leu Val 500 505 510 Met Thr Phe Val Ile Thr Arg Ile Leu Arg Val Val Glu Arg Lys Met 515 520 525 Asp Gly Pro Ser Ala Tyr Val Lys Val Glu Glu Leu Thr Glu Glu Gly 530 535 540 Lys Glu Ser 545 11 485 PRT Staphylococcus aureus 11 Met Lys Cys Leu Ile Arg Phe Ile Leu Val Leu Gly Leu Leu Ile Ser 1 5 10 15 Ser Ala Met Val Tyr Ile Asn Pro Thr Ala His Ala Glu Gln Asp Gln 20 25 30 Thr Trp Glu Lys Ile Lys Glu Arg Gly Glu Leu Arg Val Gly Leu Ser 35 40 45 Ala Asp Tyr Ala Pro Met Glu Phe Glu His Thr Val Asn Gly Lys Thr 50 55 60 Glu Tyr Ala Gly Val Asp Ile Asp Leu Ala Lys Lys Ile Ala Lys Asp 65 70 75 80 Asn Asn Leu Lys Leu Lys Ile Val Asn Met Ser Phe Asp Ser Leu Leu 85 90 95 Gly Ala Leu Lys Thr Gly Lys Ile Asp Ile Ile Ile Ser Gly Met Thr 100 105 110 Ser Thr Pro Glu Arg Lys Lys Gln Val Asp Phe Ser Asp Ser Tyr Met 115 120 125 Met Thr Lys Asn Ile Met Leu Val Lys Lys Asp Lys Val Asn Glu Tyr 130 135 140 Lys Asp Ile Lys Asp Phe Asn Asn Lys Lys Val Gly Ala Gln Lys Gly 145 150 155 160 Thr Glu Gln Glu Lys Ile Ala Gln Thr Glu Ile Glu Asn Ala Ser Ile 165 170 175 Thr Ser Leu Ser Arg Leu Pro Asp Val Ile Leu Ala Leu Lys Ser Gly 180 185 190 Lys Val Glu Gly Ala Val Val Glu Lys Pro Val Ala Glu Ala Tyr Leu 195 200 205 Lys Gln Asn Pro Lys Leu Gly Ile Ser Asn Val Lys Phe Asn Glu Glu 210 215 220 Glu Lys Asp Thr Val Ile Ala Val Pro Lys Asp Ser Pro Lys Leu Leu 225 230 235 240 Ser Gln Ile Asn Lys Thr Ile Lys Glu Val Lys Asp Lys Gly Leu Ile 245 250 255 Asp Lys Tyr Met Thr Asn Ala Ala Asn Ala Met Asn Asp Asp Ser Gly 260 265 270 Phe Ile Ser Lys Tyr Gly Ser Phe Phe Leu Lys Gly Ile Lys Ile Thr 275 280 285 Ile Leu Ile Ser Leu Ile Gly Val Ala Leu Gly Ser Ile Leu Gly Ala 290 295 300 Phe Val Ala Leu Met Lys Leu Ser Lys Ile Lys Ile Ile Ser Trp Ile 305 310 315 320 Ala Ser Ile Tyr Ile Glu Ile Leu Arg Gly Thr Pro Met Leu Val Gln 325 330 335 Val Phe Ile Val Phe Phe Gly Ile Thr Ala Ala Leu Gly Leu Asp Ile 340 345 350 Ser Ala Leu Val Cys Gly Thr Ile Ala Leu Val Ile Asn Ser Ser Ala 355 360 365 Tyr Ile Ala Glu Ile Ile Arg Ala Gly Ile Asn Ala Val Asp Lys Gly 370 375 380 Gln Met Glu Ala Ala Arg Ser Leu Gly Leu Asn Tyr Arg Gln Thr Met 385 390 395 400 Lys Ser Val Ile Met Pro Gln Ala Ile Lys Asn Ile Leu Pro Ala Leu 405 410 415 Gly Asn Glu Phe Val Thr Leu Ile Lys Glu Ser Ser Ile Val Ser Thr 420 425 430 Ile Gly Val Gly Glu Ile Met Phe Asn Ala Gln Val Val Gln Gly Ile 435 440 445 Ser Phe Asp Pro Phe Thr Pro Leu Ile Val Ala Ala Ala Leu Tyr Phe 450 455 460 Val Leu Thr Phe Val Leu Thr Arg Ile Met Asn Met Ile Glu Gly Arg 465 470 475 480 Leu Asn Ala Ser Asp 485 12 485 PRT Staphylococcus epidermidis 12 Met Lys Cys Leu Phe Lys Met Leu Ser Ile Ile Ile Ile Met Leu Ser 1 5 10 15 Thr Phe Thr Leu Phe Ile Ser Pro Ser Thr Tyr Ala Asn Glu Asp Glu 20 25 30 Asn Trp Thr Lys Ile Lys Asn Arg Gly Glu Leu Arg Val Gly Leu Ser 35 40 45 Ala Asp Tyr Ala Pro Leu Glu Phe Glu Lys Thr Ile His Gly Lys Thr 50 55 60 Glu Tyr Ala Gly Val Asp Ile Glu Leu Ala Lys Lys Ile Ala Lys Asp 65 70 75 80 Asn His Leu Lys Leu Lys Ile Val Asn Met Gln Phe Asp Ser Leu Leu 85 90 95 Gly Ala Leu Lys Thr Gly Lys Ile Asp Ile Ile Ile Ser Gly Met Thr 100 105 110 Thr Thr Pro Glu Arg Lys Lys Glu Val Asp Phe Thr Lys Pro Tyr Met 115 120 125 Ile Thr Asn Asn Val Met Met Ile Lys Lys Asp Asp Ala Lys Arg Tyr 130 135 140 Gln Asn Ile Lys Asp Phe Glu Gly Lys Lys Ile Ala Ala Gln Lys Gly 145 150 155 160 Thr Asp Gln Glu Lys Ile Ala Gln Thr Glu Ile Glu Asp Ser Lys Ile 165 170 175 Ser Ser Leu Asn Arg Leu Pro Glu Ala Ile Leu Ser Leu Lys Ser Gly 180 185 190 Lys Val Ala Gly Val Val Val Glu Lys Pro Val Gly Glu Ala Tyr Leu 195 200 205 Lys Gln Asn Ser Glu Leu Thr Phe Ser Lys Ile Lys Phe Asn Glu Glu 210 215 220 Lys Lys Gln Thr Cys Ile Ala Val Pro Lys Asn Ser Pro Val Leu Leu 225 230 235 240 Asp Lys Leu Asn Gln Thr Ile Asp Asn Val Lys Glu Lys Asn Leu Ile 245 250 255 Asp Gln Tyr Met Thr Lys Ala Ala Glu Asp Met Gln Asp Asp Gly Asn 260 265 270 Phe Ile Ser Lys Tyr Gly Ser Phe Phe Ile Lys Gly Ile Lys Asn Thr 275 280 285 Ile Leu Ile Ser Leu Val Gly Val Val Leu Gly Ser Ile Leu Gly Ser 290 295 300 Phe Ile Ala Leu Leu Lys Ile Ser Lys Ile Arg Pro Leu Gln Trp Ile 305 310 315 320 Ala Ser Ile Tyr Ile Glu Phe Leu Arg Gly Thr Pro Met Leu Val Gln 325 330 335 Val Phe Ile Val Phe Phe Gly Thr Thr Ala Ala Leu Gly Leu Asp Ile 340 345 350 Ser Ala Leu Ile Cys Gly Thr Ile Ala Leu Val Ile Asn Ser Ser Ala 355 360 365 Tyr Ile Ala Glu Ile Ile Arg Ala Gly Ile Asn Ala Val Asp Lys Gly 370 375 380 Gln Thr Glu Ala Ala Arg Ser Leu Gly Leu Asn Tyr Arg Gln Thr Met 385 390 395 400 Gln Ser Val Val Met Pro Gln Ala Ile Lys Lys Ile Leu Pro Ala Leu 405 410 415 Gly Asn Glu Phe Val Thr Leu Ile Lys Glu Ser Ser Ile Val Ser Thr 420 425 430 Ile Gly Val Ser Glu Ile Met Phe Asn Ala Gln Val Val Gln Gly Ile 435 440 445 Ser Phe Asp Pro Phe Thr Pro Leu Leu Val Ala Ala Leu Leu Tyr Phe 450 455 460 Leu Leu Thr Phe Ala Leu Thr Arg Val Met Asn Phe Ile Glu Gly Arg 465 470 475 480 Met Ser Ala Ser Asp 485 13 5 PRT Staphylococcus aureus MISC_FEATURE (3)..(3) X = Any amino acid 13 Leu Pro Xaa Thr Gly 1 5 14 22 PRT Staphylococcus aureus 14 Ala Leu Lys Thr Gly Lys Ile Asp Ile Ile Ile Ser Gly Met Thr Ser 1 5 10 15 Thr Pro Glu Arg Lys Lys 20 15 18 PRT Staphylococcus aureus 15 Val Glu Gly Ala Val Val Glu Lys Pro Val Ala Glu Ala Tyr Leu Lys 1 5 10 15 Gln Asn 16 15 PRT Staphylococcus aureus 16 Glu Tyr Ala Gly Val Asp Ile Asp Leu Ala Lys Lys Ile Ala Lys 1 5 10 15 

What is claimed is:
 1. An isolated amino acid sequence having a sequence selected from the group consisting of ALKTGKIDIIISGMTSTPERKK (SEQ ID NO: 14), VEGAVVEKPVAEAYLKQN (SEQ ID NO: 15), and EYAGVDIDLAKKIAK (SEQ ID NO: 16) and capable of generating an antibody that can recognize an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO:
 16. 2. An isolated surface protein from gram-positive bacteria which includes the sequence according to claim
 1. 